THE

TOTAL

STRUCTURE

OF

TETSUO Department

THE

ANTIBIOTIC

SHIBA and

of Chemistry,

YASUO MUKUNOKI

Faculty

Toyonaka,

LONGICATENAMYCIN*

of Science,

Osaka,

Osaka

University,

560, Japan

The peptide antibiotic longicatenamycin produced by a Streptomyces strain S-520 is a complex mixture of several congeners. The determination of the total structure of longicatenamycin was performed on the mixture using the procedure of N-bromosuccinimide oxidation followed by EDMAN degradation. The structure of the most abundant congener of longicatenamycin can be represented as cyclo (glycyl-L-2-amino6-methylheptanoyl-D-valyl-D-ornithyl-threo-48-hydroxy-L-glutamyl-5-chloro-D-tryptophyl) (Fig. 1: 1=4, m=0, n=3). There are three amino acid positions in which variations are found. The antibiotic complex contains compounds in which L-2-amino-6-methylheptanoic acid is replaced with either L-2-amino-5-methylhexanoic acid or L-2-amino7-methyloctanoic acid; further D-valine with D-isoleucine as well as n-ornithine with

A new antibiotic was isolated from the culture filtrate of Streptomyces diastaticus strain S-520 by SHOJI et al. in 1970.1) With their consent, we termed this antibiotic longicatenamycin, name of which

was derived from the unique and characteristic

unusually long aliphatic Gram-positive

side chain.

Longicatenamycin

possesses antibacterial

In our previous papers, all constituent hydroxy-L-glutamic hydroxyglutamic the structures

acid, L-2-amino-6-methylheptanoic

D-ornithine

acid (2) (or L-2-amino-5-methylhexanoic

acid (3)) and

5-chloro-D-tryptophan.

acid and the amino acids 1, 2, 3 are new

naturally

was first found in the hydrolyzate of longicatenamycin

The amino acids mentioned in the Darentheses above are the alternate components instance,

D-valine sponds

the

within

as the

2

I

or

Consequently, complex *

This

October

the molar

ratio to

is found

homologous 3

in

the

of

sum of

glycine

corre-

error

occurring

amino

acids,

amino peptide

work

presented

was

Fig.

1.

Total

structure

of longicatenamycin.

acid

with and

molecule. could

of

in our recent study.2,3) residues found in more

to one.

exchanged

longicatenamycin composed

longi-

of the

the experimental

mixture

1974.

Among them, i -

complex.

hydrolyzate

D-isoleucine

D-ornithine

D-lysine with

of the antibiotic

in

and

Similarly,

(or D-lysine), threo-(3-

of which were proposed by SHOJI et al.4,5) and confirmed by us synthetically. 2,3)

5-Chlorotryptophan

catenamycin

against

amino acids of this antibiotic were fully clarified.'2,3)

D-valine (or D-isoleucine),

acid (1), or L-2-amino-7-methyloctanoic

For

activity

bacteria.1)

It contains one mole of glycine,

minor

the

presence of amino acids with

be a

2 x 2 x 3 =12

at the

18th Symposium

on

the

Chemistry

of Natural

Products

, Kyoto,

562

THE

congeners maximally. microorganisms. Determination

JOURNAL

Such occurrence

OF

ANTIBIOTICS

is rather common

AUG.

in peptide

of the primary structure of peptide antibiotics

by sequential analysis on a pure congener. closely related peptides was extremely current distribution

antibiotics

1975

produced

by

usually has been carried out

In this case, however,

separation

difficult, since an efficient separation

of the

many

method like counter

was not readily applicable due to the very poor solubility of the complex

in most organic solvents except lower alcohol and dimethyl sulfoxide, and the presence of the acid

labile 5-chlorotryptophan.

Therefore,

in this study,

we attempted

to carry

out

the

sequential analysis of amino acids using a mixture of the congeners without separation. Longicatenamycin, derivative

as a mixture, was 2,4-dinitrophenylated

which showed principally

hydrolyzate

to give a 2,4-dinitrophenyl

a single spot on a thin-layer chromatogram.

of DNP-longicatenamycin,

only ornithine

(or lysine) disappeared

(DNP)

In the acid in amino acid

analysis and ninhydrin-positive DNP-amino acids were detected at the same time. These DNP amino acids were neutral on electrophoresis and insoluble in ether. These were compared with synthetic a-DNP-ornithine layer chromatography.

(or a-DNP-lysine) Although,

and d-DNP-ornithine

the corresponding

derivatives

(or s-DNP-lysine)

by thin-

of both amino acids were not

separated sufficiently, a- and (o-isomers were distinguished satisfactorily. The DNP-amino acids in the hydrolyzate were identical to 6-DNP-ornithine (or s-DNP-lysine). This fact suggested that longicatenamycin

is a cyclic hexapeptide

the (o-amino function

in the ornithine

In order to determine catenamycin,

in which a single free amino group is ascribed to

(or lysine) residue.

the amino acid sequence, i.e., the total primary

partial hydrolysis in 6 N hydrochloric

structure

of longi-

acid was carried out at 110'C for 1.5 hours

in an evacuated tube. The hydrolyzate was separated by paper electrophoresis.

3-Hydroxyglutamic

acid was detected in an acidic part, and glycine as well as 5-chlorotryptophan

in a neutral

part, all as free amino acids. From a basic part a tripeptide, composed of the residual amino acids, i.e., 2 (or 1, 3), valine (or isoleucine) and ornithine (or lysine), was isolated. From the result of a subtractive

EDMMAN degradation

sequence 2(1, 3)-Val(Ile)-Orn(Lys)

on this tripeptide,

could be deduced.

In this experiment

acids belonging to the same pair showed similar behaviors ratios.

This indicates that the simultaneous

as shown in Table 1, the partial the homologous

keeping nearly

the original

sequential analysis using the complex

amino molar

mixture

of

many congeners proceeded successfully in this case. With a view to obtain other applied

to effect partial

ii) N hydrochloric

acid, room Table

Proposed

peptide

hydrolysis,

1.

sequence

Composition

e.g.,

temperature,

fragments,

2

0.42

0.54

1st Cycle

0.11

2nd Cycle

hydrochloric

conditions

of the

basic

70'C,

tripeptide.

3)

(Val

Ile)

(Orn, Lys)

0.15

0.92 0.80 0.25

0.30 0.26

1.00 1.00 1.00

3rd Cycle 1: L-2-Amino-5-methylhexanoic 7-methyloctanoic acid.

acid;

2: L-2-Amino-6-methylheptanoic

were

acid, 37~C, 24 hours,

7 days, iii) 2 N sodium hydroxide,

EDMAN degradation (1

the following various

i) concentrated

acid;

3: L-2-Amino-

8 days,

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NO.

Table Proposed

8

2.

THE

Subtractive

sequence

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EDMAN degradation

(Gly) 1.2 0.32 t

Composition 1st Cycle 2nd Cycle

2

(1 0.35

0.55

0.30

0.45

OF

ANTIBIOTICS

of the

3)

(Val

0.24 0.11

0.64 0.70 0.72 0.21

t

3rd Cycle 4th

Cycle

5th

Cycle

1: L-2-Amino-5-methylhexanoic octanoic acid; HyGlu: threoTable Proposed

3.

sequence

peptide

563

obtained

by NBS

oxidation.

Ile) (Orn, Lys) (HyGlu) (ClTrp) 0.36 0.3C 0.28 t

1.2 0.75 0.87 1.00 0.20

0.46 0.41 0.56 0.55 1.00

acid; 2: L-2-Amino-6-methylheptanoic acid; 3: L-2-Amino-7-methyl-Hydroxy-L-glutamic acid; ClTrp: 5-Chloro-n-tryptophan; t: trace.

Recovered

(Gly)

amino

(1

acids

from 2

PTH

fractions

3)

(Val

in EDMAN degradation.

Ile) (Orn, Lys) (HyGlu) (C1Trp)

1st Cycle 2 nd Cycle 3rd Cycle 4th Cycle 5th Cycle

0.36 t

0.50

0.15

t 0.84

0.16

t t t ++a

1: L-2-Amino-5-methylhexanoic acid; 2: L-2-Amino-6-methylheptanoic acid; 3: L-2-Amino-7-methyloctanoic acid; HyGlu: threo-B-Hydroxyglutamic acid; ClTrp: 5-Chloro-n-tryptophan; t: trace; a): detected as PTH amino acid.

iv) N sodium hydroxide, 42°C, 24 hours, v) N sodium hydroxide, 37°C, 6 days, vi) acetic acidwater (1:1), 100°C, 24 hours etc., all in evacuated tubes. However, in every case, no peptide fragment other than the tripeptide described above was obtained. Furthermore, several proteolytic enzymes, including thermolysin which is known to have the specificity for cleavage at amino side of hydrophobic amino acid residues, were inactive against this cyclic peptide. In order to elucidate the complete structure, selective chemical cleavage at carboxyl side of 5-chlorotryptophan by N-bromosuccinimide oxidation was attempted next.6) Oxidation of longicatenamycin with excess N-bromosuccinimide gave a single ninhydrin-positive product. This result is an additional evidence for the cyclic peptide structure of longicatenamycin. The reaction process could be followed by disappearance of absorption maximum at 280 nm due to indole nucleus and the appearance of a new maximum at 320 nm. The linear peptide obtained by N-bromosuccinimide oxidation was purified by Sephadex G-10 column and then subjected to EDMANdegradation in the usual manner. Peptides produced on successive degradations were analyzed by amino acid analysis after complete acid hydrolysis. The result is summarized in Table 2. The lower value for i3-hydroxyglutamic acid in the original composition of the peptide is due to the partial decomposition of this amino acid during acid hydrolysis.3) The phenylthiohydantoyl (PTH) amino acid produced on each step of EDMAN degradation was hydrolyzed with hydrochloric acid and the recovered amino acid was analyzed as shown in Table 3. In the case of P-hydroxyglutamic acid, detection of PTH-amino acid by thin-layer chromatography was employed in order to circumvent the problem of the acid catalysed decomposition. Again in the degradation the homologous amino acids in each pair reacted analogously throughout all of the procedures. From these sequential analyses, the total structure of longicatenamycin was established as

THE

564

depicted

in Fig. 1.

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In this structure,

OF

hydrophobic

ANTIBIOTICS

characteristic that

structural

the tripeptide

acid are linked in another

amino acids like ornithine

side of the cyclic molecule.

where glycine, (3-hydroxyglutamic

tryptophan

fact

amino acids was resistant to the partial acid hydrolysis

acid and

5-chlorotryptophan

were liberated

acids. SHOJI et al. observed two spots i.e., LCM-I and LCM-II on thin-layer DNP-longicatenamycin.7)

(or This

feature may relate to the biological activity and the experimental

with hydrophobic

1975

amino acids like 2 (or 1, 3) and valine (or

isoleucine) are located in neighboring positions, while hydrophilic lysine) and (3-hydroxyglutamic

AUG.

as free amino

chromatogram

of

However after we had found the existence of acid-sensitive 5-chloro-

as a new component,2,3) SHOJI et al. reinvestigated

without extensive exposure to acidic media.

the isolation

of the antibiotic

The sample thus obtained gave single spot on tlc

with either a DNP or an acetyl derivative. Additionally, in view of the fact that LCM-II was converted to LCM-I with acid and reverse change never occurred even in alkaline solution, LCM-I seems to be an artifact attributed

to the acid degradation

of 5-chlorotryptophan

residue

in crude longicatenamycin. From NMR study of the temperature dependence of NH protons in longicatenamycin containing all congeners, one of six CO-NH protons was found to participate in an intramolecular hydrogen bond. separation

However, an exact assignment of the NMR signals must wait for the complete

of congeners. Experimental

All melting points are uncorrected. For amino acid analysis, Hitachi Amino Acid Analyzer KLA-3 was employed. Acidic and neutral amino acids were analyzed on 50-cm column using 0.2 N citric acid buffer at pH 4.25 except 2-amino-7-methyloctanoic acid (3) and 5-chlorotryptophan for which 15-cm column and 0.2 N citric acid buffer at pH 5.28 were used. Paper electrophoresis was carried out in a pyridine-acetic acid-water (30:4:966) buffer solution on Toyo filter paper No. 51. 2,4-Dinitrophenyllongicatenamycin. To a mixed solution of longicatenamycin (200 mg) in methanol and aqueous sodium hydrogencarbonate (400 mg), was added a methanolic solution (20 ml) of 1-fluoro-2,4-dinitrobenzene (2 g). The reaction mixture was stirred at room temperature in the dark for 2 hours. A yellow precipitate was filtered and reprecipitated from hot methanol and water. This precipitation was repeated three times; yield 150 mg; mp 256-261°C (decomp.). a-2,4-Dinitrophenyl-L-ornithine. 8-Benzyloxycarbonyl-L-ornithine (0.5 g) prepared through the copper complex in the usual manner8) was suspended in a mixture of water (10 ml) and ethanol (5 ml). An aqueous solution (5 ml) of sodium hydrogencarbonate (0.2 g) and an ethanolic solution (20 ml) of 1-fluoro-2,4-dinitrobenzene (0.3 g) were added to the suspension. The reaction mixture was stirred at room temperature in the dark for 2 hours. After acidification with N hydrochloric acid, it was extracted with ethyl ether. The extract was washed with water, dried with magnesium sulfate and then evaporated in vacuo. The residue was dissolved in acetic acid (10 ml) and treated with hydrogen bromide in acetic acid (5 ml) at room temperature for 1.5 hours. To the reaction mixture, water was added and the resulting solution was washed with ethyl ether. The solution was passed through Dowex 50 (H+ form) column and a-DNP-ornithine was eluted with N ammonia. The eluate was evaporated in vacuo to obtain an oily product.9) a-2,4-Dinitrophenyl-L-lysine. In a similar manner to that of the ornithine derivative, aDNP-L-lysine was prepared from s-benzyloxycarbonyl-L-lysine. 8-2,4-Dinitrophenyl-L-ornithine. Crystals of i5-DNP-L-ornithine

hydrochloride

(360 mg) was

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THE

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ANTIBIOTICS

565

obtained from L-ornithine hydrochloride (500mg) through the copper complex according to the procedure of F. SANGER,9) mp 215-218'C (decomp.). Found: C, 39.11; H, 4.57; N, 16.45; Cl, 10.37%. Calcd. for C11H14,O6N2.HCl: C, 39.47; H, 4.52; N, 16.74; Cl, 10.59%. s-2,4-Dinitrophenyl-L-lysine. This compound was prepared in a similar manner to that of o-DNP-L-ornithine. Hydrolysis of 2,4-Dinitrophenyllongicatenamycin. DNP-longicatenamycin (25 mg) was hydrolyzed with 6 N hydrochloric acid (3 ml) at 110°C for 24 hours. The DNP-amino acid formed showed a positive ninhydrin reaction and was insoluble in ethyl ether. It moved as a neutral amino acid on paper electrophoresis. Rf values on thin-layer chromatogram on developing with methyl ethyl ketone-ethyl ether (3:2) were: DNP-amino acid obtained by the hydrolysis, 0.20; synthetic a-DNP-ornithine (or a-DNP-lysine), 0.10; synthetic o-DNP-ornithine (or s-DNP-lysine) 0.20. Partial Hydrolysis of Longicatenamycin. Longicatenamycin was partially hydrolyzed with 6 N hydrochloric acid in an evacuated tube at 110°C for 1.5 hours. The hydrolyzate was subjected to paper electrophoresis and each band was cut off and extracted with a mixture of water and acetic acid (1: 1). (3-Hydroxyglutamic acid was detected in an acidic part on tlc, and glycine and 5-chlorotryptophan in a neutral part. From a basic part, a single spot of a peptide was detected on tic, Rf: 0.42 (n-butanol -acetic acid-water, 4:1:2); 0.15 (methyl ethyl ketone-pyridine-water-acetic acid, 35:5:5:1). The subtractive EDMANdegradation of this basic peptide was carried out by the usual procedure.10) The result is given in Table 1. N-Bromosuccinimide Oxidation of Longicatenamycin. To a solution of longicatenamycin (100 mg, 0.12 mM) in a mixture of acetic acid (4 ml) and methanol (4 ml), was added Nbromosuccinimide (178 mg, 1 mM) in methanol (4 ml) and the mixture was allowed to stand at room temperature for 50 minutes. After decomposition of excess N-bromosuccinimide with formic acid (2 ml) for 30 minutes, the reaction mixture was concentrated below 35'C. The syrup thus obtained was dissolved in water, and was subjected to chromatography on a Sephadex G-10 column (1.5x 50 cm) with water elution. Fractions showing UV absorption at 320 nm were combined and concentrated in vacuo. Lyophilization produced a white powder, yield 70 mg. This linear peptide was subjected to EDMANdegradation. In each step, the phenylthiohydantoyl (PTH) amino acid obtained was hydrolyzed with 6 N hydrochloric acid at 135°C for 12 hours to recover free amino acid which was analyzed by amino acid analyzer. PTH-phydroxyglutamic acid obtained in the final degradation step was detected by comparison with authentic sample on tlc, Rf: 0.10 (chloroform-methanol, 9:1); 0.12 (chloroform-methanolwater 45:12:1); 0.65 (chloroform-methanol-acetic acid, 15:5:1); 0.44 (n-butanol saturated with water). Furthermore, the residual peptides of each degradation were analyzed after complete hydrolysis with 6 N hydrochloric acid at 110°C for 24 hours. Acknowledgement The Ltd. for antibiotic.

authors their

are kind

deeply

indebted

supply

of

to Dr. HIDEO OTSUKA and

longicatenamycin

and

their

Dr.

consent

JUNICHI SHOJI to

our

of

structural

Shionogi study

Co., on

this

References

1) SHOJI, J.; S. KOZUKI, M. MAYAMA & N. SHIMAOKA: A new peptide antibiotic complex S-520. I. Isolation and characterization. J. Antibiotics 23: 429-431, 1970 2) SHIBA, T.; Y. MUKUNOKI & H. AKIYAMA: Isolation and characterization of a new amino acid, 5-chloro-D-tryptophan from antibiotic longicatenamycin. Tetrahedron Letters 1974: 3085-3086, 1974 3) SHIBA, T.; Y. MUKUNOKI & H. AKIYAMA: Component amino acids of the antibiotic longicatenamycin. Isolation of 5-chloro-D-tryptophan. Bull. Chem. Soc. Japan 48: 1902--.1906, 1975 4) SHOJI, J. & R. SAKAZAKI: A new peptide antibiotic complex S-520. III. Isolation of L-threo-;3hydroxyglutamic acid from the hydrolyzate. J. Antibiotics 23: 418-419, 1970

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5) SHOJI, J. & R. SAKAZAKI: A new peptide antibiotic complex S-520. IV. Isolation of three new amino acids from the hydrolyzate, J. Antibiotics 23: 519-521, 1970 6) RAMACHANDRAN,L. K. & B. WITKOP: Selective cleavage of C-tryptophyl peptide bonds in proteins and peptides. J. Amer. Chem. Soc. 81: 4028-4032, 1959 7) SHOJI, J. & R. SAKAZAKI: A new antibiotic complex S-520. II. Further characterization and degradative studies. J. Antibiotics 23: 432-436, 1970 8) SYNGE, R. L. M.: The synthesis of some dipeptides related to gramicidin S.Biochem. J. 42: 99104, 1948 9) SANGER, F.: The free amino group of gramicidin S. Biochem. J. 40: 261-262, 1946 10) KONIGSBERG,W. & R. J. HILL: Structure of human hemoglobin. III. The sequence of amino acids in the tryptic peptides of the a chain. J. Biol. Chem. 237: 2547-2561, 1962